Histone post-translational modifications (PTMs), chromatin-remodeling enzymes, DNA methylation, and histone chaperones play critical roles in the organization and control of our genomes. Importantly, disruption or mutation of the proteins or enzymes responsible for chromatin regulation underlies a number of human diseases, most notably cancer. Our long-term goal is to elucidate the molecular underpinnings of chromatin regulation and define how this regulation contributes to human biology and disease. Our lab has spearheaded the pursuit of this goal, making seminal discoveries that explain how the chromatin-modifying machinery ?writes? and ?reads? histone PTMs and how histone PTMs function in transcription, DNA replication, DNA repair, and maintenance of DNA methylation. Yet, despite considerable progress, there remain many crucial knowledge gaps. For example, it is largely unknown the extent to which histone PTMs function in a combinatorial manner. Similarly, we do not fully understand the biochemical basis for recognition of histone PTMs by the many different reader domains in chromatin- associated proteins nor do we fully understand how paired reader domains bind nucleosomes in a multivalent manner. Finally, we do not know the functions of newly identified histone PTMs, e.g., lysine crotonylation. With this MIRA award, our lab will close these knowledge gaps by answering three of the foremost fundamental questions in chromatin biology: 1) How do histone-modifying enzymes and chaperones regulate transcription, the cell cycle, and maintain chromatin integrity? 2) How do recently discovered histone PTMs such as crotonylation contribute to gene regulation? 3) To what extent do histone PTMs collaborate to regulate downstream functions in chromatin?